Mapping the colorectal tumor microbiota.

The gut microbiome in patients with colorectal cancer (CRC) is different than that of healthy controls. Previous studies have profiled the CRC tumor microbiome using a single biopsy. However, since the morphology and cellular subtype vary significantly within an individual tumor, the possibility of sampling error arises for the microbiome within an individual tumor. To test this hypothesis, seven biopsies were taken from representative areas on and off the tumor in five patients with CRC. The microbiome composition was strikingly similar across all samples from an individual. The variation in microbiome alpha-diversity was significantly greater between individuals' samples then within individuals. This is the first study, to our knowledge, that shows that the microbiome of an individual tumor is spatially homogeneous. Our finding strengthens the assumption that a single biopsy is representative of the entire tumor, and that microbiota changes are not limited to a specific area of the neoplasm.

Electrically induced muscle cramps induce hypertrophy of calf muscles in healthy adults.

OBJECTIVES: Skeletal muscles usually cramp at short lengths, where the tension that can be exerted by muscle fibers is low. Since high tension is an important anabolic stimulus, it is questionable if cramps can induce hypertrophy and strength gains. In the present study we investigated if electrically induced cramps (EIMCs) can elicit these adaptations. METHODS: 15 healthy male adults were randomly assigned to an intervention (IG; n=10) and a control group (CG; n=5). The cramp protocol (CP) applied twice a week to one leg of the IG, consisted of 3x6 EIMCs, of 5 s each. Calf muscles of the opposite leg were stimulated equally, but were hindered from cramping by fixating the ankle at 0° plantar flexion (nCP). RESULTS: After six weeks, the cross sectional area of the triceps surae was similarly increased in both the CP (+9.0±3.4%) and the nCP (+6.8±3.7%). By contrast, force of maximal voluntary contractions, measured at 0° and 30° plantar flexion, increased significantly only in nCP (0°: +8.5±8.8%; 30°: 11.7±13.7%). CONCLUSION: The present data indicate that muscle cramps can induce hypertrophy in calf muscles, though lacking high tension as an important anabolic stimulus.

The repeated bout effect: is the blunted creatine kinase response an effect of an altered enzyme inactivation kinetic?

AIM: If unaccustomed lengthening contractions are repeated within a certain period of time, muscle damage symptoms are blunted. This observation, often referred to as the repeated bout effect (RBE), also holds true for the response of muscle damage markers like creatine kinase (CK). However, measuring plasma enzyme activity rather than the concentration of enzyme protein might conceal the actual amount of damaged tissue. Therefore, the primary aim of the study was to investigate if the RBE of CK can partially be explained by enzyme inactivation. METHODS: Ten healthy male subjects performed two bouts of 100 drop-to-vertical jumps (DVJs) from a 70-cm high platform at an interval of three weeks. CK activity, CK concentration, and neutrophils were measured prior to, and on four consecutive days after the interventions. RESULTS: Besides significant main effects, there was a significant group by time interaction for the specific CK activity (CK activity in blood [U/L] divided by the enzyme concentration [ng/mL]). Higher values following the first bout (133.1±99.4 U/µg) than the second bout (94.7±63.0 U/µg) indicate that the ratio of inactive to active CK molecules increased. Neutrophil levels were similar following both bouts and differed only at 8 hours (7.0±2.5 bout 1, 5.1±1.6 bout 2). CONCLUSION: The findings of the present study support the hypothesis that the blunted response of CK activity after a repeated bout of eccentric exercise is not solely the result of tissue protection, but can be at least partially attributed to enzyme inactivation.

Preclinical characterisation of the GM-CSF receptor as a therapeutic target in rheumatoid arthritis.

OBJECTIVE: Previous work has suggested that the granulocyte macrophage colony stimulating factor (GM-CSF)-GM-CSF receptor α axis (GM-CSFRα) may provide a new therapeutic target for the treatment of rheumatoid arthritis (RA). Therefore, we investigated the cellular expression of GM-CSFRα in RA synovial tissue and investigated the effects of anti-GM-CSFRα antibody treatment in vitro and in vivo in a preclinical model of RA. METHODS: We compared GM-CSFRα expression on macrophages positive for CD68 or CD163 on synovial biopsy samples from patients with RA or psoriatic arthritis (PsA) to disease controls. In addition, we studied the effects of CAM-3003, an anti-GM-CSFR antibody in a collagen induced arthritis model of RA in DBA/1 mice. The pharmacokinetic profile of CAM-3003 was studied in naïve CD1(ICR) mice (see online supplement) and used to interpret the results of the pharmacodynamic studies in BALB/c mice. RESULTS: GM-CSFRα was expressed by CD68 positive and CD163 positive macrophages in the synovium, and there was a significant increase in GM-CSFRα positive cells in patients in patients with RA as well as patients with PsA compared with patients with osteoarthritis and healthy controls. In the collagen induced arthritis model there was a dose dependent reduction of clinical arthritis scores and the number of F4/80 positive macrophages in the inflamed synovium after CAM-3003 treatment. In BALB/c mice CAM-3003 inhibited recombinant GM-CSF mediated margination of peripheral blood monocytes and neutrophils. CONCLUSIONS: The findings support the ongoing development of therapies aimed at interfering with GM-CSF or its receptor in various forms of arthritis, such as RA and PsA.

Motor point map of upper body muscles.

PURPOSE: The purpose of the present study was to systematically investigate the upper body motor point (MP) positions of selected muscles and to create an atlas of the identified MPs. METHODS: MPs were searched bilaterally in 15 male and 15 female subjects by scanning the skin with a special pen electrode at low stimulation frequency (3 Hz) and current amplitude (<10 mA). The following muscles were investigated: biceps brachii, triceps brachii, deltoideus, trapezius, latissimus dorsi, erector spinae (lumbar part), pectoralis minor and major, and rectus abdominis. RESULTS: A total of 1,563 MPs were identified. The MPs could be clustered into 31 distinct positions on each side of the body. However, the number of MPs per muscle varied between subjects: 2 MPs were found for the biceps brachii, 2-3 for the triceps brachii, 4-5 for the deltoideus, 2-3 for the pectoralis major, 1 MP for the pectoralis minor, 4-5 for the trapezius, 3-4 for the latissimus dorsi, 4-5 for the rectus abdominis, and 2-3 for the erector spinae in its lumbar part. Referring to the applied grid, upper limb and lower back muscles presented a low inter-individual variation, whereas MPs of the deltoideus, the pectoralis major, and the rectus abdominis were characterized by a poor homogeneity. All MPs were found to be highly symmetrical between both sides of the body (r = 0.96; p < 0.001). CONCLUSION: The presented data and the corresponding map will help physiotherapists, and conditioning specialists improve their neuromuscular electrical stimulation therapy or training, respectively.

Protein engineering and preclinical development of a GM-CSF receptor antibody for the treatment of rheumatoid arthritis.

BACKGROUND AND PURPOSE: For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen 'sink' effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients. EXPERIMENTAL APPROACH: We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development. KEY RESULTS: Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg(-1) and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg(-1) dosing. CONCLUSIONS AND IMPLICATIONS: This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.

Neonatal intensive care unit: predictive models for length of stay.

OBJECTIVE: Hospital length of stay (LOS) is important to administrators and families of neonates admitted to the neonatal intensive care unit (NICU). A prediction model for NICU LOS was developed using predictors birth weight, gestational age and two severity of illness tools, the score for neonatal acute physiology, perinatal extension (SNAPPE) and the morbidity assessment index for newborns (MAIN). STUDY DESIGN: Consecutive admissions (n=293) to a New England regional level III NICU were retrospectively collected. Multiple predictive models were compared for complexity and goodness-of-fit, coefficient of determination (R (2)) and predictive error. The optimal model was validated prospectively with consecutive admissions (n=615). Observed and expected LOS was compared. RESULT: The MAIN models had best Akaike's information criterion, highest R (2) (0.786) and lowest predictive error. The best SNAPPE model underestimated LOS, with substantial variability, yet was fairly well calibrated by birthweight category. LOS was longer in the prospective cohort than the retrospective cohort, without differences in birth weight, gestational age, MAIN or SNAPPE. CONCLUSION: LOS prediction is improved by accounting for severity of illness in the first week of life, beyond factors known at birth. Prospective validation of both MAIN and SNAPPE models is warranted.

Persistent perforation in non-faeculant diverticular peritonitis--incidence and clinical significance.

BACKGROUND: Non-resectional strategies (NRS) have improved outcomes for a sub-group of patients with perforated diverticulitis. NRS are applicable to patients with non-faeculant peritonitis (Hinchey II and III). Success is dependent on the initial perforation sealing, which Hinchey estimated occurred 'most of the time'. An exact percentage remains ill-defined. OBJECTIVE: We aimed to define the percentage and clinical significance of a persistent perforation in non-faeculant diverticular peritonitis. DESIGN: A retrospective review was conducted of all patients admitted with a diagnosis of perforated diverticulitis between January 1999 and July 2010. Patients undergoing an emergency operation were analysed according to Hinchey and physiological and operative severity scores and compared with histological findings. RESULTS: One hundred fifteen patients were identified. Fifty-three patients underwent a 'resectional' procedure. At surgery, 15 patients had faeculent peritonitis, 27 patients had purulent peritonitis and 11 patients had a contained abscess. Of the patients with non-faeculant peritonitis, 2/9 (22.2 %) Hinchey II and 10/27 (37.1 %) Hinchey III patients had persistent perforation on review of histology. Persistent perforation was associated with a significant increase in morbidity, length of stay, physiological and operative severity score (p = 0.015, 0.011, 0.049 and 0.002, respectively). CONCLUSION: A proportion of patients with non-faeculant peritonitis have a persistent perforation which is associated with a poorer outcome and is likely to result in failure of a non-resectional management strategy. Updated classification systems and tailored peri-operative investigations are required to identify this sub-group of patients and improve patient outcomes.

Changes in outcome and management of perforated diverticulitis over a 10 year period.

INTRODUCTION: Aggressive non-operative intervention and evolving surgical strategies have altered the treatment of perforated diverticulitis in the acute setting. These strategies have predominantly been implemented over the last decade. The aim of this study was to assess the impact of this on patient outcome during their index admission and subsequently. METHODS: Consecutive patients admitted with acute diverticulitis between 1999 and 2010 were identified. Patient demographics, treatment strategies and outcomes were collected and analysed. Patients who had an episode of perforated diverticulitis during their index admission were followed. RESULTS: 739 patients were admitted with acute diverticulitis. Of these, 115 (15.7%) had perforated diverticulitis. 53 (47.8%) underwent an intervention. There was a reduction in the mean age of patients admitted with acute diverticulitis of 8.9% over the study period (p = 0.002). There was a significant increase in the use of CT scanning pre-operatively (p < 0.001). 'Non-resectional' interventions have emerged in the form of laparoscopic lavage (n = 5) and percutaneous abscess drainage (n = 14). There was associated improved length of stay (p < 0.001). CONCLUSION: Outcomes for patients with perforated diverticulitis have improved, contributed to in part by an increased use of non-resectional management strategies.

Enhanced recovery after elective colorectal surgery: now the standard of care.

Enhanced recovery programmes have been studied in randomised trials with evidence of quicker recovery of gut function, reduced morbidity, mortality and hospital stay and improved physiological and nutritional outcomes. They aim to reduce the physiological and psychological stress of surgery and consequently the uncontrolled stress response. The key elements, reduced pre-operative fasting, intravenous fluid restriction and early feeding after surgery, are in conflict with traditional management plans but are supported by strong clinical evidence. Given the strength of the current data enhanced recovery should now be the standard of care.

An unusual presentation of Fournier's gangrene.

INTRODUCTION: Fournier's gangrene is a rare severe necrotising fasciitis of the genitalia. CASE: A case of Fournier's gangrene caused by perforated sigmoid diverticulitis in a patient with systemic lupus erythematosus is presented along with a review of the relevant literature.

Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.

BACKGROUND AND PURPOSE: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications. EXPERIMENTAL APPROACH: An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies. KEY RESULTS: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions. CONCLUSIONS: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.

Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Rectal cancer.

Surgery is the cornerstone of rectal cancer treatment. Oncological cure and overall survival continue to be the main goals, but sparing of the anal sphincter mechanism and functional results are also important. The modern management of rectal cancer is a multidisciplinary approach, and pre-operative staging is of crucial importance when planning treatment in these patients. Pre-operative staging is used to determine the indication for neoadjuvant therapy prior to surgical resection or to determine whether local excision is an option in carefully selected patients with early rectal cancer. Surgery in the form of total mesorectal excision (TME) has become the standard of care for mid and distal rectal cancers. Early rectal cancers do not require neoadjuvant therapy. For locally advanced cancers of the lower two-thirds of the rectum, the combination of surgical resection with chemoradiotherapy decreases local recurrence rates and probably improves overall survival. Whereas in the past local excision was only contemplated in patients who were unfit for radical surgery or for local palliation in cases of metastatic disease, over the last number of years there has been increasing interest in local treatment with curative intent in early rectal cancer.

Transforming growth factor-beta1 is the predominant isoform required for breast cancer cell outgrowth in bone.

Transforming growth factor (TGF)-beta signaling is a potent modulator of the invasive and metastatic behavior of breast cancer cells. Indeed, breast tumor responsiveness to TGF-beta is important for the development of osteolytic bone metastases. However, the specific TGF-beta isoforms that promote breast cancer outgrowth in bone is unknown. We demonstrate that expression of a TGF-beta ligand trap, which neutralizes TGF-beta1 and TGF-beta3, in MDA-MB-231 breast cancer cells diminished their outgrowth in bone and reduced the severity of osteolytic lesion formation when compared with controls. We further show that a reduction or loss of TGF-beta1 expression within the bone microenvironment of TGF-beta1+/- and TGF-beta1-/- mice significantly reduced the incidence of breast tumor outgrowth compared with wild-type animals. Interestingly, those tumors capable of growing within the tibiae of TGF-beta1-deficient mice had upregulated expression of all three TGF-beta isoforms. Finally, breast cancer cells expressing the TGF-beta ligand trap showed a pronounced reduction in their ability to form osteolytic lesions when injected into the tibiae of TGF-beta1+/- mice. Thus, our studies show that both host- and tumor-derived TGF-beta expression plays a critical role during the establishment and outgrowth of breast cancer cells in bone.

Cancer systems biology: exploring cancer-associated genes on cellular networks.

Genomic alterations lead to cancer complexity and form a major hurdle for comprehensive understanding of the molecular mechanisms underlying oncogenesis. In this review, we describe recent advances in studying cancer-associated genes from a systems biology point of view. The integration of known cancer genes onto protein and signaling networks reveals the characteristics of cancer genes within networks. This approach shows that cancer genes often function as network hub proteins which are involved in many cellular processes and form focal nodes in information exchange between many signaling pathways. Literature mining allows constructing gene-gene networks, in which new cancer genes can be identified. The gene expression profiles of cancer cells are used for reconstructing gene regulatory networks. By doing so, genes which are involved in the regulation of cancer progression can be picked up from these networks, after which their functions can be further confirmed in the laboratory.

Sandwich ELISA detection of Clostridium perfringens cells and alpha-toxin from field cases of necrotic enteritis of poultry.

Sandwich ELISAs (sELISAs) for the detection of Clostridium perfringens cells and alpha-toxin were developed and used to screen intestinal samples from normal broiler chickens and from clinical cases of necrotic enteritis. The assays clearly distinguished between the two sets of samples. The sELISA absorbance values from samples obtained from the majority of healthy birds were low and those from the majority of necrotic enteritis cases were high. Together, the assays provide a suitable test for the rapid screening for the diagnosis of necrotic enteritis in poultry.

Substrate cleavage by caspases generates protein fragments with Smac/Diablo-like activities.

Smac/Diablo and HtrA2/Omi promote apoptosis by binding to and antagonizing IAP proteins, including the 'X chromosome-linked inhibitor of apoptosis' (XIAP). Here we show that caspase-mediated proteolysis of a limited subset of cell death substrates exposes functional Smac/Diablo-like N-termini after cleavage, which are able to bind to and antagonize XIAP. We propose that this mechanism may establish a feedforward sensitization of the apoptotic pathway and contribute to the functional redundancy of IAP antagonism. In addition, this may be particularly relevant in Alzheimer's disease since the caspase-generated C31 peptide, an established cytotoxin, acquires Smac/Diablo-like properties after apoptotic processing.